The objective is to produce new imaging probes and associated protocols for preclinical biomedical research and for clinical use in humans. The first specific aim is to discover small synthetic molecules that selectively target anionic cell membrane surfaces with exposed phosphatidylserine and distinguish them from the near-neutral membrane surfaces of healthy cells. One application is cell death imaging, as a method to detect disease and measure treatment efficacy. Preliminary work has shown that a family of fluorescent zinc coordination complexes can act as optical imaging probes for dead and dying tissue in various cell and animal models. The selectivity over healthy tissue is very high, even though binding affinities for exposed phosphatidylserine are moderate (low micromolar dissociation constants). To improve cell targeting performance for clinical applications, a systematic cycle of synthesis and screening will be employed to find next-generation compounds that have 500-fold higher binding affinity for phosphatidylserine. This will enable microdosing, which is the key to successful translation. The imaging ability of lead compounds will be evaluated in cell culture and animal models of cell death with a focus on producing the first set of practically useful, optical imaging probes for non-invasive assessment of the efficacy of anticancer therapies. The second specific aim is to create a new paradigm for planar optical imaging of tumors using near-infrared chemiluminescent and fluorescent (CLF) nanoparticles. The chemiluminescence is thermally-activated (that is, no chemical or electrical stimulus is needed) which means that the nanoparticles can be stored at low temperature and they only become chemiluminescent when warmed to body temperature. Preliminary results in mice show that chemiluminescence imaging permits identification of target sites that are more than two centimeters below the animal surface, which is about five times deeper than currently achieved using planar fluorescence imaging. The new imaging paradigm will be demonstrated by conducting experiments using relatively deep-tissue animal models of spontaneous prostate cancer and orthotopic colon cancer. Chemiluminescence imaging will be used to locate the tumor tissue that is targeted by the nanoparticles, and fluorescence microscopy of thin histology sections taken from the same specimen will quantify uptake into the tumor parenchyma. The power of this new imaging technology will be employed to answer questions concerning the ability of the tumor vascular-penetrating peptide iRGD to promote nanoparticle entry into tumors.